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Supplementary Figure S1. Vistusertib (AZD2014) causes a decrease in RB phosphorylation in the absence of cell cycle arrest. Supplementary Figure S2. Different mTOR inhibitors in combination with palbociclib cause a decrease in cell viability. Supplementary Figure S3. The effects of vistusertib and palbociclib in the CTC-174 model in vivo. Supplementary Figure S4. Analysis of cell cycle and cell morphology. Supplementary Figure S5. The effects of vistusertib (AZD2014) and palbociclib on P21 expression levels. Supplementary Figure S6. Analysis of apoptosis in vitro and in vivo. Supplementary Figure S7. Robust inhibition of mTOR signalling by short-term treatment with RAD001 or vistusertib (AZD2014) does not affect ER binding. Supplementary Table S1. Selected genes and primer IDs. Supplementary Table S2. Supplementary Table S3. Palbociclib resistant cell lines are sensitive to vistusertib (AZD2014).

Supplementary Table 1. Mass Spectrometer and UPLC system parameters, Supplementary Table 2. Optimization parameters for mass spectrometry analysis, Supplementary Table 3. Estimated mouse PK model parameter values for AZD2811, Supplementary Table 4. Summary table of the pIC50 and nuclear area, Supplementary Table 5: PK parameters summary, Supplementary Figure 1. Representative bioluminescent images of the disseminated U2932Luc2 DLBCL xenograft model in vivo taken at day 18 post treatment initiation, Supplementary Figure 2. 96h post treatment, AZD2811 nanoparticle treatment reduces pHH3 levels with no evidence of polyploidy in the OCI-LY19 xenograft model in vivo, Supplementary Figure 3. In SCID mice AZD2811 nanoparticle is well tolerated at the different doses and schedules explored

The cyclin dependent kinase (CDK)–retinoblastoma (RB)–E2F pathway plays a critical role in the control of cell cycle in estrogen receptor–positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor–resistant cell lines reactivate the CDK–RB–E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908–20. ©2018 AACR.

Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1–blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell–mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.

Supplementary Figure 1. Pharmacokinetic (PK) properties of AZD3458 in mouse. Supplementary Figure 2. In vivo pharmacodynamic properties of AZD3458 in mouse neutrophils. Supp Fig 3. PI3K-�/mTOR pathway regulates expression of IL-10/IL-12 via LPS-induced glycolytic burst. Supplementary Figure 4. AZD3458 improves checkpoint activity in CT-26 tumor models. Supplementary Figure 5. AZD3458 improves checkpoint activity in 4T1 tumor models. Supplementary Figure 6. Top canonical IPA pathways across tissues in AZD3458+PD-1 combination group. Supplementary Figure 7. Graphical abstract.

Supplementary Materials and Methods. Suplementary Table 1. Tumor Immuno-phenotyping. Suplementary Table 2. Flow cytometry panels.

Barasertib (AZD1152), a pro-drug of the highly potent and selective Aurora B kinase inhibitor AZD2811, showed promising clinical activity in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients administered as a 4-day infusion. To improve potential therapeutic benefit of Aurora B kinase inhibition, a nanoparticle formulation of AZD2811 has been developed to address limitations of repeated intravenous infusion. One of the challenges with the use of nanoparticles for chronic treatment of tumors is optimizing dose and schedule required to enable repeat administration to sustain tumor growth inhibition. AZD2811 gives potent cell growth inhibition across a range of DLBCL cells lines in vitro. In vivo, repeat administration of the AZD2811 nanoparticle gave antitumor activity at half the dose intensity of AZD1152. Compared with AZD1152, a single dose of AZD2811 nanoparticle gave less reduction in pHH3, but increased apoptosis and reduction of cells in G1 and G2–M, albeit at later time points, suggesting that duration and depth of target inhibition influence the nature of the tumor cell response to drug. Further exploration of the influence of dose and schedule on efficacy revealed that AZD2811 nanoparticle can be used flexibly with repeat administration of 25 mg/kg administered up to 7 days apart being sufficient to maintain equivalent tumor control. Timing of repeat administration could be varied with 50 mg/kg every 2 weeks controlling tumor control as effectively as 25 mg/kg every week. AZD2811 nanoparticle can be administered with very different doses and schedules to inhibit DLBCL tumor growth, although maximal tumor growth inhibition was achieved with the highest dose intensities.

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.

Peripheral T-cell lymphoma (PTCL) represents a rare group of heterogeneous diseases in urgent need of effective treatments. A scarcity of disease-relevant preclinical models hinders research advances. Here, we isolated a novel mouse (m)PTCL by serially transplanting a lymphoma from a germinal center B-cell hyperplasia model (Cγ1-Cre Blimp1fl/fl ) through immune-competent mice. Lymphoma cells were identified as clonal TCRβ+ T-helper cells expressing T-follicular helper markers. We also observed coincident B-cell activation and development of a de novo B-cell lymphoma in the model, reminiscent of B-cell activation/lymphomagenesis found in human PTCL. Molecular profiling linked the mPTCL to the high-risk "GATA3" subtype of PTCL, showing GATA3 and Th2 gene expression, PI3K/mTOR pathway enrichment, hyperactivated MYC, and genome instability. Exome sequencing identified a human-relevant oncogenic β-catenin mutation possibly involved in T-cell lymphomagenesis. Prolonged treatment responses were achieved in vivo by targeting ATR in the DNA damage response (DDR), a result corroborated in PTCL cell lines. This work provides mechanistic insight into the molecular and immunological drivers of T-cell lymphomagenesis and proposes DDR inhibition as an effective and readily translatable therapy in PTCL.

Purpose: Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action. Experimental Design: Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen. Results: Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti–PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti–PD-L1. Conclusions: STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

BackgroundThe Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3, which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered, unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man.MethodsWe have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons.ResultsAZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs, reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO, which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs, reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo, strongly modulated Treg effector molecules (eg, ICOS, CTLA-4, CD25 and 4-1BB), and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy.ConclusionsAntisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669).

In a phase 1b study of acalabrutinib (a covalent Bruton tyrosine kinase (BTK) inhibitor) in combination with vistusertib (a dual mTORC1/2 inhibitor) in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), multiple ascending doses of the combination as intermittent or continuous schedules of vistusertib were evaluated. The overall response rate was 12% (3/25). The pharmacodynamic (PD) profile for acalabrutinib showed that BTK occupancy in all patients was >95%. In contrast, PD analysis for vistusertib showed variable inhibition of phosphorylated 4EBP1 (p4EBP1) without modulation of AKT phosphorylation (pAKT). The pharmacokinetic (PK)/PD relationship of vistusertib was direct for TORC1 inhibition (p4EBP1) but did not correlate with TORC2 inhibition (pAKT). Cell-of-origin subtyping or next-generation sequencing did not identify a subset of DLBCL patients with clinical benefit; however, circulating tumor DNA dynamics correlated with radiographic response. These data suggest that vistusertib does not modulate targets sufficiently to add to the clinical activity of acalabrutinib monotherapy. Clinicaltrials.gov identifier: NCT03205046.

Immunotherapy has transformed cancer treatment by promoting durable clinical responses in a proportion of patients; however, treatment still fails in many patients. Innate immune cells play a key role in the response to immunotherapy. Crosstalk between innate and adaptive immune systems drives T-cell activation but also limits immunotherapy response, as myeloid cells are commonly associated with resistance. Hence, innate cells have both negative and positive effects within the tumor microenvironment (TME), and despite investment in early clinical trials targeting innate cells, they have seen limited success. Suppressive myeloid cells facilitate metastasis and immunotherapy resistance through TME remodeling and inhibition of adaptive immune cells. Natural killer (NK) cells, in contrast, secrete inflammatory cytokines and directly kill transformed cells, playing a key immunosurveillance role in early tumor development. Myeloid and NK cells show reciprocal crosstalk, influencing myeloid cell functional status or antigen presentation and NK effector function, respectively. Crosstalk between myeloid cells and the NK immune network in the TME is especially important in the context of therapeutic intervention. Here we discuss how myeloid and NK cell interactions shape anti-tumor responses by influencing an immunosuppressive TME and how this may influence outcomes of treatment strategies involving drugs that target myeloid and NK cells.

BackgroundChemotherapy and targeted agent anti-cancer efficacy is largely dependent on the proliferative state of tumours, as exemplified by agents that target DNA synthesis/replication or mitosis. As a result, cell cycle specificities of a number of cancer drugs are well known. However, they are yet to be described in a quantifiable manner.MethodsA scalable cell synchronisation protocol used to screen a library of 235 anti-cancer compounds exposed over six hours in G1 or S/G2 accumulated AsPC-1 cells to generate a cell cycle specificity (CCS) score.FindingsThe synchronisation method was associated with reduced method-related cytotoxicity compared to nocodazole, delivering sufficient cell cycle purity and cell numbers to run high-throughput drug library screens. Compounds were identified with G1 and S/G2-associated specificities that, overall, functionally matched with a compound's target/mechanism of action. This annotation was used to describe a synergistic schedule using the CDK4/6 inhibitor, palbociclib, prior to gemcitabine/AZD6738 as well as describe the correlation between the CCS score and published synergistic/antagonistic drug schedules.InterpretationThis is the first highly quantitative description of cell cycle-dependent drug sensitivities that utilised a tractable and tolerated method with potential uses outside the present study. Drug treatments such as those shown to be G1 or S/G2 associated may benefit from scheduling considerations such as after CDK4/6 inhibitors and being first in drug sequences respectively.FundingCancer Research UK (CRUK) Institute core grants C14303/A17197 and C9545/A29580. The Li Ka Shing Centre where this work was performed was generously funded by CK Hutchison Holdings Limited, the University of Cambridge, CRUK, The Atlantic Philanthropies and others.

The heterogeneity of breast cancer plays a major role in drug response and resistance and has been extensively characterized at the genomic level. Here, a single-cell breast cancer mass cytometry (BCMC) panel is optimized to identify cell phenotypes and their oncogenic signalling states in a biobank of patient-derived tumour xenograft (PDTX) models representing the diversity of human breast cancer. The BCMC panel identifies 13 cellular phenotypes (11 human and 2 murine), associated with both breast cancer subtypes and specific genomic features. Pre-treatment cellular phenotypic composition is a determinant of response to anticancer therapies. Single-cell profiling also reveals drug-induced cellular phenotypic dynamics, unravelling previously unnoticed intra-tumour response diversity. The comprehensive view of the landscapes of cellular phenotypic heterogeneity in PDTXs uncovered by the BCMC panel, which is mirrored in primary human tumours, has profound implications for understanding and predicting therapy response and resistance., The heterogeneity of breast cancer has a major role in drug response and resistance. In this study, the authors use patient-derived tumour xenografts as a platform for drug testing and correlation with single-cell proteomic phenotypes characterized by mass cytometry.

Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1–blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell–mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.

The genomic complexity and heterogeneity of high-grade serous ovarian cancer (HGSOC) has hampered the realisation of successful therapies and effective personalised treatment is an unmet clinical need. Here we show that primary HGSOC spheroid models can be used to predict drug response and use them to demonstrate that somatic copy number alterations (SCNAs) in frequently amplified HGSOC cancer genes significantly correlate with gene expression and drug response. These genes are often located in areas of the genome with frequent clonal SCNAs. MYC chromosomal copy number is associated with ex-vivo and clinical response to paclitaxel and ex-vivo response to mTORC1/2 inhibition. Activation of the mTOR survival pathway in the context to MYC-amplified HGSOC is mostly due to increased prevalence of SCNAs in genes from the PI3K pathway. These results suggest that SCNAs encompassing driver genes could be used to inform therapeutic response in the context of clinical trials testing personalised medicines.

Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, thus perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells. Translocations involving the Mll1 (Kmt2a) gene generate powerful oncogenic fusion proteins, predominantly affecting infant and paediatric AML and ALL patients. The early stages of leukaemogenic transformation are typically inaccessible from human patients and conventional mouse models. Here, we take advantage of cells conditionally blocked at the multipotent haematopoietic progenitor stage to develop a MLL-r model capturing early cellular and molecular consequences of MLL-ENL expression based on a clear clonal relationship between parental and leukaemic cells. Through a combination of scRNA-seq, ATAC-seq and genome-scale CRISPR-Cas9 screening, we identify pathways and genes likely to drive the early phases of leukaemogenesis. Finally, we demonstrate the broad utility of using matched parental and transformed cells for small molecule inhibitor studies by validating both previously known and other potential therapeutic targets., The oncogene MLL is frequently translocated in leukemia, resulting in oncogenic fusion proteins. Here, the authors report a temporally controlled mouse model of MLL-ENL driven leukemia AND identify therapeutic targets associated with early MLL-ENL driven leukaemogenesis.

The PI3Kα signaling pathway is frequently hyper-activated in breast cancer (BrCa), as a result of mutations/amplifications in oncogenes (e.g. HER2), decreased function in tumor suppressors (e.g. PTEN) or activating mutations in key components of the pathway. In particular, activating mutations of PIK3CA (~45%) are frequently found in luminal A BrCa samples. Genomic studies have uncovered inactivating mutations in MAP3K1 (13-20%) and MAP2K4 (~8%), two upstream kinases of the JNK apoptotic pathway in luminal A BrCa samples. Further, simultaneous mutation of PIK3CA and MAP3K1 are found in ~11% of mutant PIK3CA tumors. How these two alterations may cooperate to elicit tumorigenesis and impact the sensitivity to PI3K and AKT inhibitors is currently unknown. Using CRISPR gene editing we have genetically disrupted MAP3K1 expression in mutant PIK3CA cell lines to specifically create in vitro models reflecting the mutational status of PIK3CA and MAP3K1 in BrCa patients. MAP3K1 deficient cell lines exhibited ~2.4-fold increased proliferation rate and decreased sensitivity to PI3Kα/δ(AZD8835) and AKT (AZD5363) inhibitors (~2.61 and ~5.23-fold IC50 increases, respectively) compared with parental control cell lines. In addition, mechanistic analysis revealed that MAP3K1 disruption enhances AKT phosphorylation and downstream signaling and reduces sensitivity to AZD5363-mediated pathway inhibition. This appears to be a consequence of deficient MAP3K1-JNK signaling increasing IRS1 stability and therefore promoting IRS1 binding to p85, resulting in enhanced PI3Kα activity. Using 3D-MCF10A-PI3KαH1047R models, we found that MAP3K1 depletion increased overall acinar volume and counteracted AZD5363-mediated reduction of acinar growth due to enhanced proliferation and reduced apoptosis. Furthermore, in vivo efficacy studies revealed that MAP3K1-deficient MCF7 tumors were less sensitive to AKT inhibitor treatment, compared with parental MCF7 tumors. Our study provides mechanistic and in vivo evidence indicating a role for MAP3K1 as a tumor suppressor gene at least in the context of PIK3CA-mutant backgrounds. Further, our work predicts that MAP3K1 mutational status may be considered as a predictive biomarker for efficacy in PI3K pathway inhibitor trials.

Background The ability to modulate immune-inhibitory pathways using checkpoint blockade antibodies such as αPD-1, αPD-L1, and αCTLA-4 represents a significant breakthrough in cancer therapy in recent years. This has driven interest in identifying small-molecule-immunotherapy combinations to increase the proportion of responses. Murine syngeneic models, which have a functional immune system, represent an essential tool for pre-clinical evaluation of new immunotherapies. However, immune response varies widely between models and the translational relevance of each model is not fully understood, making selection of an appropriate pre-clinical model for drug target validation challenging. Methods Using flow cytometry, O-link protein analysis, RT-PCR, and RNAseq we have characterized kinetic changes in immune-cell populations over the course of tumor development in commonly used syngeneic models. Results This longitudinal profiling of syngeneic models enables pharmacodynamic time point selection within each model, dependent on the immune population of interest. Additionally, we have characterized the changes in immune populations in each of these models after treatment with the combination of α-PD-L1 and α-CTLA-4 antibodies, enabling benchmarking to known immune modulating treatments within each model. Conclusions Taken together, this dataset will provide a framework for characterization and enable the selection of the optimal models for immunotherapy combinations and generate potential biomarkers for clinical evaluation in identifying responders and non-responders to immunotherapy combinations.

Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of malignancies with an aggressive clinical course. Molecular and immunophenotypic analysis have linked PTCL subgroups to derivation from differentiated T cells, including T follicular helper (Tfh), Th1, and Th2 cells. Effective therapies for these PTCLs are undefined and tumor models are limited. We developed a novel mouse model of PTCL and applied it to evaluate responses to therapy. An in vivo transplantable cell line was isolated by passaging a splenic lymphoma arising from a mouse with B cell-specific Blimp1-deletion (Cγ1-Cre Blimp1fl/fl) into C57bl/6J mice. Lymphomas were profiled by flow cytometry, immunohistochemistry, RNA, and exome sequencing. Mice intravenously inoculated with lymphoma cells developed disseminated disease affecting primarily lymphoid tissues and the liver. Model latency to welfare endpoint was 3-4 weeks. Transplantable cells expressed multiple T-cell lineage markers, were CD8 negative and CD4 positive, but gradually downregulated CD4 with passaging. Lymphoma cells (“mPTCL”) also expressed a single TCRvβ chain, suggesting origin from a T helper cell clone. Immunophenotypically, mPTCL stably expressed Tfh markers ICOS, PD-1, CD40L, and low BCL6, and thus was potentially classifiable as a surrogate PTCL of Tfh phenotype. However, the transcriptome of purified mPTCL or spleens indicated a resemblance to Th2 cells and corresponding PTCL-GATA3 subtype (a PTCL-not otherwise specified). In accord with Tfh and Th2 helper function, B cells in the tumor microenvironment had an activated phenotype but were nonexpansive. Investigation into oncogenic pathways in mPTCL revealed a predicted deleterious mutation in Ctnnb1 (β-catenin) and enrichment for Myc-activated, cell cycle regulation, and DNA damage response genes. In vivo, mPTCL progression was significantly and potently delayed by monotherapy inhibition of ataxia telangiectasia and Rad3-related (ATR), a kinase central to resolving replication stress. Taken together, we established a novel model of PTCL with overlapping Tfh and Th2 features. Our findings in this model suggest the use of a DNA damage response inhibitor as a potential treatment strategy for aggressive PTCL-GATA3 lymphomas. Citation Format: Elizabeth Kuczynski, Giulia Morlino, Alison Peter, Dinis Calado, Charles Sinclair, Larissa Carnevalli. Molecular characterization of a mouse model of peripheral T-cell lymphoma with Tfh and Th2 features [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-20.

PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kδ modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. PI3K inhibitors as a class and PI3Kδ specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835, a dual PI3Kα/δ inhibitor, specifically on the tumor immune microenvironment using syngeneic models. Continuous suppression of PI3Kα/δ was not required for anti-tumor activity, as tumor growth inhibition was potentiated by an intermittent dosing/schedule in vivo. Moreover, PI3Kα/δ inhibition delivered strong single agent anti-tumor activity, which was associated with dynamic suppression of T-regs, improved CD8+ T-cell activation and memory in mouse syngeneic tumor models. Strikingly, AZD8835 promoted robust CD8+ T-cell activation dissociated from its effect on T-regs. This was associated with enhancing effector cell viability/function. Together these data reveal novel mechanisms by which PI3Kα/δ inhibitors interact with the immune system and validate the clinical compound AZD8835 as a novel immunoncology drug, independent of effects on tumor cells. These data support further clinical investigation of PI3K pathway inhibitors as immuno-oncology agents.

The cyclin dependent kinase (CDK)-retinoblastoma (RB)-E2F pathway plays a critical role in the control of cell cycle in estrogen receptor-positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor-resistant cell lines reactivate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR., CRUK Cambridge Institute, Cambridge, UK (Jason Carroll, Igor Chernukhin, Rasmus Siersbæk3) Novo Nordisk Fonden (NNF 14136) (Rasmus Siersbæk)

Barasertib (AZD1152), a pro-drug of the highly potent and selective Aurora B kinase inhibitor AZD2811, showed promising clinical activity in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients administered as a 4-day infusion. To improve potential therapeutic benefit of Aurora B kinase inhibition, a nanoparticle formulation of AZD2811 has been developed to address limitations of repeated intravenous infusion. One of the challenges with the use of nanoparticles for chronic treatment of tumors is optimizing dose and schedule required to enable repeat administration to sustain tumor growth inhibition. AZD2811 gives potent cell growth inhibition across a range of DLBCL cells lines in vitro. In vivo, repeat administration of the AZD2811 nanoparticle gave antitumor activity at half the dose intensity of AZD1152. Compared with AZD1152, a single dose of AZD2811 nanoparticle gave less reduction in pHH3, but increased apoptosis and reduction of cells in G1 and G2–M, albeit at later time points, suggesting that duration and depth of target inhibition influence the nature of the tumor cell response to drug. Further exploration of the influence of dose and schedule on efficacy revealed that AZD2811 nanoparticle can be used flexibly with repeat administration of 25 mg/kg administered up to 7 days apart being sufficient to maintain equivalent tumor control. Timing of repeat administration could be varied with 50 mg/kg every 2 weeks controlling tumor control as effectively as 25 mg/kg every week. AZD2811 nanoparticle can be administered with very different doses and schedules to inhibit DLBCL tumor growth, although maximal tumor growth inhibition was achieved with the highest dose intensities.

Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state., This work was supported by the FOR2033 and SFB873 funded by the Deutsche Forschungsgemeinschaft (DFG), the Dietmar Hopp Foundation (all to A.T.), and the Wellcome Trust (to A.S.).

Objectives: PI3Kγ inhibition re-polarizes macrophages to an immuno-stimulatory phenotype, thereby activating a T-cell mediated tumor immune response. AZD3458 is a highly selective PI3Kγ inhibitor. Administration of AZD3458 in combination with checkpoint inhibitors such as α-PD-(L)1 antibodies had greater anti-tumor effects (TGI 26-86%) than checkpoint inhibitor alone in 4T1, LLC, CT-26 and MC-38 syngeneic mouse models. In these, AZD3458 remodeled the tumor microenvironment (TME), reducing immunosuppressive markers (e.g in 4T1 model there was a 20% decrease in total macrophages and 50% decrease in markers of immune suppression like CD206 by flow cytometry) and promoting cytotoxic T-cell activation (e.g. in CT-26 model there was a 2-fold increase in gzmB mRNA). We developed a predictive quantitative systems pharmacology (QSP) model, to quantitatively simulate TME effects and delineate mechanistic principles underlying AZD3458 and α-PD-(L)1 synergistic effects. Methods: The QSP model captures mechanistic, molecular and cellular interactions between PI3Kγ inhibition and checkpoint inhibitors, together with the pharmacokinetics acting on the respective targets. Features such as PI3Kγ inhibition-dependent tumor-associated macrophages, protein expression of immunosuppressive markers, reduction of MDSC activation and promotion of cytotoxic T-cell activation were included in the model. These immuno-changes were then linked to tumor cell death, resulting in macroscopic dynamic effects on tumor size. Some model parameters were taken from the literature and internal studies; some were estimated using NLME modeling of tumor size data. Results: The model adequately described individual and population tumor size patterns. Inter-animal variability was described using a random effect on a parameter related to the ability of T cells to infiltrate the tumor in response to systemic antigen. Additionally, the model incorporated in one quantitative framework data from 4 syngeneic tumors capturing respective changes in TME conditions. Simulations for the various treatments supported the mechanistic interpretation of the observed AZD3458 and α-PD-(L)1 synergistic effects. The model was further used to simulate treatment scenarios, to infer optimal dosing and scheduling for the combination and given underlying TME conditions. Conclusions: This study provides quantitative mechanistic insights into the links between PI3Kγ inhibition and anti-tumor immune responses, supporting our understanding of how AZD3458 may alleviate brakes in a myeloid immuno-suppressive TME and revert resistance to immunotherapy. This mechanistic understanding is critical when proceeding with dose escalation in an early clinical trial setting, as it allows to contextualize any potential compound-induced immuno-modulation in patients, for given doses and schedules. Citation Format: Pablo Morentin Gutierrez, Yuri Kosinsky, Kirill Peskov, Ivan Azarov, Lulu Chu, Veronika Voronova, Martin Johnson, Yingxue Chen, Larissa Carnevalli, Danielle Carroll, Michele Moschetta, Teresa Klinowska, Gabriel Helmlinger. Mechanistic insights and dose optimization for AZD3458, a novel selective PI3Kg immuno-modulator, using a quantitative systems approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 104.

The PI3Kγ isoform is a key regulator of immune cell proliferation, survival, migration and activation. PI3Kγ inhibitors have the potential to treat a variety of diseases including respiratory, inflammatory, metabolic disorders and cancer. In the context of cancer PI3Kγ inhibition re-polarizes macrophages to an immuno-stimulatory phenotype activating a T-cell mediated tumor immune response. Selective PI3Kγ inhibitors have potential as monotherapy and in combination with checkpoint inhibition to overcome resistance and to enhance efficacy of checkpoint inhibitors. AZD3458 is a highly selective PI3Kγ clinical candidate kinase inhibitor with a cellular IC50 of 8nM with a 100-fold selectivity over PI3Kδ. In vitro, AZD3458 inhibits pAKTS308/S473 in human macrophages and mouse CD11b activation at 32nM and 30nM free IC50, respectively. At these concentrations AZD3458 reverses macrophage polarization increasing IL12/IL-10 ratio and does not impact T cell proliferation and function analysed by gene expression profiling, cytokine quantification and flow cytometry. Oral administration of AZD3458 (20mg/Kg BID) remodeled the tumor microenvironment in 4T1 orthotopic breast tumor model. AZD3458 decreased tumor associated macrophages by 20% compared to vehicle and reduced overall protein expression of immunosuppressive markers CD206 and PD-L1 measured by flow cytometry. In addition, AZD3458 reduced MDSC/neutrophil activation and promoted cytotoxic T-cell activation in vivo, measured by GzmB and Perforin mRNA and protein expression. Consistent to the observed tumor remodeling, combination with checkpoint inhibitors α-PD-1 or α-PD-L1 antibodies (10mg/kg 3x week) had greater anti-tumor effects than checkpoint inhibitor alone in 4T1, LLC, CT-26 and MC-38 mouse syngeneic models. These data demonstrate that AZD3458 can reverse myeloid suppressive tumor microenvironment and revert tumor resistance to immunotherapy in myeloid-enriched immunosuppressive tumor types. Citation Format: Larissa S. Carnevalli, Danielle Carrol, Michele Moschetta, Pablo Morentin Gutierrez, Cristina Gardelli, Molly A. Taylor, Antonio Montoya, Adina Hughes, Matthew King, Teresa Klinowska, Simon T. Barry. Novel selective PI3Kγ inhibitor AZD3458 promotes anti-tumor immune responses and reverts resistance to immunotherapy in checkpoint blockade refractory preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 100.

Metabolic adaptation to a Warburg phenotype is a hallmark of cancer, where tumors rely on glucose to produce ATP and building blocks required to support rapid tumor growth. Glycolytic metabolism requires continuous export of lactate from cells by monocarboxylate transporters (MCTs) resulting in accumulation of lactic acid in the tumor microenvironment. Tumor-derived lactate is immune-suppressive and has been shown to modify immune cell function. Inhibition of the MCT4 lactate transporter has the potential to reduce lactate in the tumor microenvironment and reverse immune suppression. AZD0095 is a highly selective, potent inhibitor of MCT4 with a cellular activity of 1-3 nM with a >1000-fold selectivity over MCT1. We confirm that lactic acid inhibits T cell proliferation and viability. AZD0095 does not inhibit T cell proliferation or survival (IC50 > 10 μM). Oral administration of AZD0095 (10-100 mg/kg BID) modulates lactate transport in mouse syngeneic models which do not express the MCT1 lactate transporter. AZD0095 treatment causes an increase in tumor-infiltrating lymphocytes and an increase in cytotoxic T cells in combination with α-PD1. AZD0095 treatment suppresses myeloid infiltration as a monotherapy and in combination with checkpoint inhibitor. Consistent with the observed changes in immune cell infiltration, the combination of AZD0095 with checkpoint inhibitors α-PD1 or α-CTLA4 (10 mg/kg 3x week) enhances the anti-tumor activity of checkpoint inhibitor monotherapy in MC-38 and EMT6 (MCT1 knock-out) mouse syngeneic models. These data demonstrate that AZD0095 can reverse lactate-driven immunosuppression and enhance response to checkpoint inhibition in pre-clinical models. Citation Format: Susan E. Critchlow, Gareth Hughes, Anna Staniszewska, Matt King, Filippos Michopoulos, Lorna Hopcroft, Martin R. Brown, Jenna Bradley, Beverley Hammond, Pablo MorentinGutierrez, Larissa Carnevalli, Elizabeth Hardaker, Frederick W. Goldberg. Reversing lactate-driven immunosuppression using the novel, potent and selective MCT4 inhibitor AZD0095 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1207.

Introduction Despite the advances in patient stratification and the understanding of basic molecular mechanisms underlining breast cancer (BC), 30% of patients with early stages of the disease later relapse. To offer more effective treatments to these patients, we need to adapt pre-clinical drug development to account for the main feature of human cancer: heterogeneity. Our group has established a biobank of 160 BC patient-derived tumour xenografts (PDTXs) and has robustly shown that PDTXs and their matching patient-derived tumour cells (PDTCs) preserve most of the originating sample’s molecular features, including intra-tumour clonal diversity (Bruna et al. Cell 2016). This integrated platform permits high-throughput (HT) assessment of drug responses ex vivo in PDTCs and their validation in vivo in PDTXs. Thus, this PDTX/PDTC biobank represents a powerful resource for pre-clinical drug development in BC. Material and methods Here, we aim to add a crucial layer of molecular characterisation at single-cell resolution by studying the phenotypic tumour heterogeneity in BC PDTXs and its role in drug response mechanisms. To achieve this goal, we employed the state-of-the-art HT single-cell method called mass cytometry (CYTOF), which combines the principles of flow cytometry and mass spectrometry allowing the simultaneous analysis of about 40 molecular events using metal-conjugated antibodies. Results and discussions We designed and validated a BC-specific panel of 35 antibodies to investigate: a)the epithelial tumour compartment; b)the stroma tumour compartment; c)oncogenic signalling; d)cell cycle and apoptosis. We used this panel in CyTOF experiments to perform an in-depth phenotypic characterisation of a representative collection of BC cell lines and molecularly distinct PDTXs. Using data-driven unsupervised methods to analyse the CyTOF data focusing on the epithelial compartment, we show that we capture different degrees of both inter- and intra-tumour functional heterogeneity. Importantly, we show that CyTOF can be used to characterise the heterogeneity in signalling dynamics within the different tumour compartments under the perturbation of therapy. Conclusion CyTOF is a novel approach to characterise heterogeneity and drug responses in BC PDTXs. The integration of high-level features, such as single-cell phenotypic profiles, and functional features, such as drug response data, obtained with CyTOF will facilitate the pre-clinical development of novel therapeutic strategies and will help to uncover mechanisms of drug resistance.

SummaryEarlier, we reported that S6K1−/− mice have reduced body fat mass, have elevated rates of lipolysis, have severely decreased adipocyte size, and are resistant to high fat diet (HFD)-induced obesity. Here we report that adipocytes of S6K1−/− mice on a HFD have the capacity to increase in size to a degree comparable to that of wild-type (WT) mice, but not in number, indicating an unexpected lesion in adipogenesis. Tracing this lesion revealed that S6K1 is dispensable for terminal adipocyte differentiation, but is involved in the commitment of embryonic stem cells to early adipocyte progenitors. We further show that absence of S6K1 attenuates the upregulation of transcription factors critical for commitment to adipogenesis. These results led to the conclusion that a lack of S6K1 impairs the generation of de novo adipocytes when mice are challenged with a HFD, consistent with a reduction in early adipocyte progenitors.

The cyclin dependent kinase (CDK) –retinoblastoma (RB) -E2F pathway plays a critical role in the control of cell cycle in estrogen receptor positive (ER+) breast cancer. Small molecule inhibitors of CDK4/6 have shown promise in this tumour type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data has suggested cooperation between the phosphatidylinositol 3-kinase (PI3K) pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F mediated transcription. We demonstrate that inhibition of mTOR using the mTORC1/2 inhibitor vistusertib at 300nM causes a >50% decrease in cyclin D1 protein levels and RB phosphorylation in three cell lines. At these concentrations, vistusertib treatment also elicits marked effects on E2F mediated transcription, causing changes in the mRNA levels of 28 out of 43 (65%) of a selected set of E2F target genes.Combined inhibition of mTOR, CDK4/6 and ER delivers profound and durable regressions in breast cancer cell lines and xenografts (110.2% tumour growth inhibition at day 48). In vivo data show, that over a period of 58 days, tumours failed to re-grow in the presence of the triplet combination compared to either agent alone, suggesting, that the triplet is necessary to maintain growth inhibition. Furthermore, we show that CDK4/6 inhibitor resistant cell lines re-activate the CDK-RB-E2F pathway, but remain sensitive to mTOR inhibition (EC50 52.7 nM in parental cells vs 39.6-73.3 nM in a number of palbociclib resistant cell populations), suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. A Phase I study (PASTOR) combining the dual TOR kinase inhibitor Vistusertib with Palbociclib, and Fulvestrant is underway to explore safety and efficacy of the triplet combination in patients with metastatic breast cancer. Citation Format: Oelmann E, Michaloglou C, Crafter C, Siersbaek R, Delpuech O, Curven J, Carnevalli L, Staniszweska A, Polanska U, Cheraghchi-Bashi A, Lawson M, Chernukhin I, McEwen R, Carroll J, Cosulich S. Combined inhibition of mTOR and CDK4/6 is required for optimal blockade of E2F function and long term growth inhibition in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-04.

In response to different cellular stresses, a family of protein kinases phosphorylates eIF2alpha (alpha subunit of eukaryotic initiation factor-2), contributing to regulation of both general and genespecific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2alpha(P) (phosphorylated eIF2alpha) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 191-194]. We show in the present study that one eIF2alpha kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2alpha(P) in these areas. In PKR-deficient animals subjected to SE, eIF2alpha phosphorylation was clearly evident coincident with activation of a secondary eIF2alpha kinase, PEK/PERK (pancreatic eIF2alpha kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2alpha phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2alpha(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2alpha contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2alpha phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.

Granzyme B is expressed by hematopoietic stem cells (HSCs) and stromal cells in response to bacterial products or chemotherapy agents and limits HSC reconstitution potential., The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4–TRIF–p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress.

Targeted deletion of S6 kinase (S6K) 1 in mice leads to higher energy expenditure and improved glucose metabolism. However, the molecular mechanisms controlling these effects remain to be fully elucidated. Here, we analyze the potential role of dietary lipids in regulating the mTORC1/S6K system. Analysis of S6K phosphorylation in vivo and in vitro showed that dietary lipids activate S6K, and this effect is not dependent upon amino acids. Comparison of male mice lacking S6K1 and 2 (S6K-dko) with wt controls showed that S6K-dko mice are protected against obesity and glucose intolerance induced by a high-fat diet. S6K-dko mice fed a high-fat diet had increased energy expenditure, improved glucose tolerance, lower fat mass gain, and changes in markers of lipid metabolism. Importantly, however, these metabolic phenotypes were dependent upon dietary lipids, with no such effects observed in S6K-dko mice fed a fat-free diet. These changes appear to be mediated via modulation of cellular metabolism in skeletal muscle, as shown by the expression of genes involved in energy metabolism. Taken together, our results suggest that the metabolic functions of S6K in vivo play a key role as a molecular interface connecting dietary lipids to the endogenous control of energy metabolism.

In this work, we show extensive phosphorylation of the alpha subunit of translation initiation factor 2 (eIF2alpha) occurring in the brain of mice subjected to 30 min of status epilepticus induced by pilocarpine. eIF2alpha(P) immunoreactivity was detected in the hippocampal pyramidal layer CA1 and CA3, cortex layer V, thalamus and amygdala. After 2 h of recovery, there was a marked decrease in total brain eIF2alpha(P), with the cortex layer V showing the most pronounced loss of anti-eIF2alpha(P) labeling, whereas the CA1 subregion had a significant increase in eIF2alpha(P). These results indicate that inhibition of protein synthesis in experimental models of epilepsy might be due to low levels of eIF2-GTP caused by the phosphorylation of eIF2alpha, and suggest that translational control may contribute to cell fate in the affected areas.

The heterotrimeric eukaryotic initiation factor (eIF) 2 binds the initiator methionyl-tRNA in a GTP-dependent mode and delivers it to the 40 S ribosomal subunit. in the present study, we have identified amino acid residues in eIF2beta required for binding to eIF2gamma in yeast. Alteration of six residues in the central region of eIF2beta abolished this interaction, as determined by GST-pull down and two-hybrid assays, and leads to cell lethality. Substitution of (131)Tyr and (132)Ser by alanine residues ((YS)-Y-131), although abolishing the binding to eIF2gamma in these assays, resulted in a functional but defective protein in vivo, imparting a temperature-sensitive growth phenotype to cells. A dramatically weakened association of this mutant protein with eIF2gamma in vivo was shown by co-immunoprecipitation. the (YS)-Y-131 mutation in eIF2beta allows translation to initiate at non-AUG codons, as defined by the ability of cells carrying an initiator codon mutation in the HIS4 mRNA to grow in the absence of histidine. the combination of this mutation with the (254)Ser --> Tyr alteration, a previously isolated suppressor of initiator codon mutations which has been shown to increase the spontaneous GTP hydrolysis in the ternary complex, caused a recessive lethality, suggesting additive defects. Thus the impaired interaction of these two subunits represents a novel type of defect in eIF2 function, providing in vivo evidence that the strength of interaction between eIF2beta and eIF2gamma defines the correct usage of the AUG codon for translation initiation. Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, Brazil Web of Science

Hyperactivity of mTORC1, a key mediator of cell growth, leads to stem cell depletion although the underlying mechanisms are poorly defined. Using spermatogonial progenitor cells (SPCs) as a model system, we show that mTORC1 impairs stem cell maintenance by a negative feedback from mTORC1 to receptors required to transduce niche-derived signals. We find that SPCs lacking Plzf, a transcription factor essential for SPC maintenance, have enhanced mTORC1 activity. Aberrant mTORC1 activation in Plzf −/− SPCs inhibits their response to GDNF, a growth factor critical for SPC self-renewal, via negative feedback at the level of the GDNF receptor. Plzf opposes mTORC1 activity by inducing expression of the mTORC1 inhibitor Redd1. Thus, we identify the mTORC1-Plzf functional interaction as a critical rheostat for maintenance of the spermatogonial pool, and propose a model whereby negative feedback from mTORC1 to the GDNF receptor balances SPC growth with self-renewal.